How to Run an mPAGE™ Protein Gel Using a Bio-Rad Electrophoresis System

1. Prepare running buffer
2. Prepare protein gel
3. Assemble electrophoresis chamber
4. Prepare and load samples
5. Run gel
6. Remove gel from cassette

*This protocol is applicable to Bio-Rad Mini-PROTEAN^® Tetra, Mini-PROTEAN II^®, and Mini-PROTEAN^® 3 electrophoresis systems.

Tips and FAQs

• Assembling a Bio-Rad Electrophoresis Chamber with mPAGE™ Gels
• Selecting MES vs. MOPS Running Buffer for Protein Gel Electrophoresis
• Sample Preparation and Loading
• How to Run a Gel
• Removing the Protein Gel

Assembling a Bio-Rad Electrophoresis Chamber with mPAGE™ Gels

1. Reverse the gaskets of the Bio-Rad running module from the standard setting (Figures 1-2). If this step is omitted, the buffer core will leak into the outer chamber.
2. Insert the mPAGE™ gel with the shorter plate facing the inner core of the chamber.

Figure 1. The rubber gasket in the Bio-Rad electrophoresis unit needs to be flipped before placing the mPAGE™ gel.

Figure 2. Left: Incorrect orientation of Bio-Rad gasket for use with mPAGE™ gels. Right: Correct orientation of Bio-Rad gasket for use with mPAGE™ gels.

Selecting MES vs. MOPS Running Buffer for Protein Gel Electrophoresis

There are two buffers that can be used as running buffers for SDS-PAGE gels: MES and MOPS. MES has a lower pKa than MOPS, which enables the gel to run faster. MES buffer gives better separation of proteins at lower molecular weights, while MOPS buffer provides better separation at higher molecular weights (Figure 3).

• Use MES running buffer to resolve small molecular weight proteins
• Use MOPS running buffer to resolve medium and high molecular weight proteins

Figure 3. Selection of MES (left image) vs. MOPS (right image) running buffer for mPAGE™ protein gel electrophoresis, based on protein size and percent acrylamide gel. Protein size for corresponding bands indicated in kDa. Smaller proteins are resolved more efficiently with MES running buffer; larger proteins are resolved more efficiently with MOPS running buffer.

Sample Preparation and Loading

1. Prepare samples just prior to electrophoresis as indicated in Table 1.

Table 1.

Reagent	Reduced sample (µL)	Non-reduced sample (µL)
Protein Sample	x	x
mPAGE™ 4X Sample Buffer	2.5	2.5
1 M DTT1	1	n/a
Deionized Water	6.5 - x	7.5 - x
Total Volume	10	10

2. Heat samples for 10 minutes at 70 °C (do not boil samples). Centrifuge samples briefly prior to loading.
3. To load the samples into the wells, vertically insert the pipette tip for optimal sample loading results (Figure 4). Do not exceed well capacity when loading samples: 80 μL for 10-well gels; 60 μL for 12-well gels; and 40 μL for 15-well gels.

Figure 4. Improper and proper pipette angle for loading samples into mPAGE™ pre-cast gels.

How to Run a Gel

1. Once the samples are loaded and buffer chambers are filled, place the cover onto the electrophoresis tank and plug the electrical leads into the power supply.
2. Run the gel at constant voltage until the dye front reaches 2 mm from the bottom of the gel cassette. Run time can vary depending on the gel percentage, running buffer, and equipment used. Refer to Table 2 for optimal voltage and typical run times best suited for the chosen gel and running buffer. When running more than one gel per electrophoresis tank, all gels should be of the same composition.

Table 2.

% Acrylamide	mPAGE™ MES SDS Running Buffer 180 V	mPAGE™ MOPS SDS Running Buffer 200 V
Typical Amperage (Starting - Ending)	Typical Run Time	Typical Amperage (Starting - Ending)	Typical Run Time
8%	109-55 mA	22 min	103-47 mA	27 min
10%	105-50 mA	26 min	97-48 mA	31 min
12%	100-46 mA	30 min	97-42 mA	34 min
4-12%	107-53 mA	26 min	97-41 mA	36 min
4-20%	106-44 mA	40 min	87-40 mA	34 min
8-16%	113-48 mA	32 min	93-44 mA	31 min

Removing the Protein Gel

1. Turn off the power supply, remove the cover from electrophoresis unit, and remove the gel.
2. Use the mPAGE™ Gel Cassette Opener to loosen the gel plates (Figure 5). The Gel Cassette Opener can be inserted between the two plastic gel plates enclosing the gel and used as a lever to separate the plates. Remember to loosen the gel plates on both sides of the gel with the cassette opener.

Figure 5.

3. Hold the gel horizontally and gently lift off the top plate.
4. Slowly peel the gel from bottom plate and place in buffer or distilled water in preparation for staining or blotting.